PROJECT SUMMARY/ABSTRACT Gut microbiota profoundly shapes immunity in humans. Dysbiosis, a disturbance in the quantity and composition of gut microbiota, leads to aberrant immune development and functioning and increases the risk of various diseases, including cancer. The effects of dysbiosis on non-gastrointestinal (non-GI) cancers are largely unknown. Moreover, the mechanistic effects of dysbiosis on endothelial-mediated immune surveillance in non-GI primary tumor microenvironment (TME) are unexplored. The goal of this proposal is to unravel the mechanisms by which dysbiosis impairs immune surveillance (via leukocyte extravasation and subsequent activation), specifically focusing on endothelial cell/stroma modulation. Our objective is to delineate the systemic influence of dysbiosis-generated cytokine profiles on endothelial-associated immune surveillance in non-GI TME. Based on our preliminary studies, our central hypothesis is that changes in cytokine expression during dysbiosis downregulate endothelial adhesion molecules (EAMs) such as ICAM-1, VCAM, and E- selectin. These EAMs are crucial for leukocytes to bind and adhere to tumor endothelial cells and transmigrate into the tumor, while therapeutics that upregulate EAMs improve extravasation. In particular, we will investigate the dysbiosis-induced modulation of EAMs in two primary non-GI tumors?melanoma and ovarian cancer?and the leukocyte extravasation enhancement effects by compounds that modulate EAMs. In the following Specific Aims we will test our hypothesis by measuring and analyzing: the induction of dysbiosis, changes in microbiota, non?tumor-EAMs and tumor-EAMs, tumor growth curves, serum cytokine levels, and leukocyte extravasation in nontumor tissues and in tumors by using our established methods (e.g., 16S rRNA sequencing, real-time imaging and in vivo flow cytometry, immunofluorescence). We will associate changes in microbiota (abundance, diversity, and composition) with the downstream effects on EAMs and leukocyte extravasation in tumor-bearing and non?tumor-bearing mice with and without cancer therapeutics. Specific Aim 1. Characterize the effects of dysbiosis on EAM expression and leukocyte-endothelial interactions in non?tumor-bearing mice. Specific Aim 2. Characterize the effects of dysbiosis on endothelial-based immune surveillance in tumors. Specific Aim 3. Enhance tumoral leukocyte extravasation by modulating EAM expression during dysbiosis. This research is significant because determining if dysbiosis contributes to tumor progression by modulating the TME will improve our understanding of microbiota and cancer crosstalk in melanoma and ovarian tumors, which could lead to novel cancer therapeutics and increase the efficacy of existing ones.